POS0625 EFFECTS OF DIFFERENTIATION STAGES OF HEALTHY AND SSc MACROPHAGES ON THE 3D HUMAN CARDIAC FIBROSIS MODEL
Authors: L. Provenzale, A. Hukara, I. Kocherova, E. Pachera, A. Laimbacher, O. Distler, P. Blyszczuk, G. Kania
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Activated cardiac fibroblasts, key regulators of cardiac fibrosis, control contraction of human fibrotic cardiac microtissues (MT)<sup>[1]</sup>. Macrophages regulate fibrotic process by interfering with the extracellular matrix (ECM) turnover. The heart is one of the primary organs affected in systemic sclerosis (SSc). <h3>Objectives</h3> To study the role of healthy donor- and SSc patient-derived macrophages in the functions of 3 dimensional (D) human cardiac MT fibrosis model. <h3>Methods</h3> CD14<sup>+</sup> blood-derived monocytes from healthy controls (HC) and SSc patients were isolated and differentiated into human monocyte-derived macrophages (hMDM) using human monocyte colony-stimulating factor (hM-CSF). A timeline of macrophage differentiation stages was performed by incubation of the CD14<sup>+</sup> cells for 1,3,5 and 7 days (d) with hM-CSF. One MT was generated by 4'000 human induced pluripotent stem cell-derived cardiomyocytes (iCM), 1'000 human cardiac fibroblasts (HCF) and 1'000 HC or SSc hMDM, mixed in a ratio 4:1:1 (HC-hMDM:iCM:HCF-MT or SSc-hMDM:iCM:HCF-MT), and formed by self-assembling in plates with ultra-low-attachment surface. Control MTs were composed with iCM/HCF in a ratio 4:1/4:2 (iCM:HCF-MT). Two days after MT assembly, the fibrotic condition was induced with transforming growth factor β1 (TGF-β1) for 10d. Contraction of each MT was assessed by recording videos, applying MUSCLEMOTION<sup>[2]</sup> and subsequent evaluation by relaxation time (ms), contraction amplitude (a.u.), contraction duration (ms), time-to-peak (ms) and beating rate (bpm). The level of fibrosis was analysed on several levels: mRNA by qPCR, ELISA to measure secreted pro-collagen-Iα1, immunohistochemistry (IHC) using Sirius Red to visualize collagen depositions and degree of fibrosis. Apoptosis was assessed using Caspase-3/7 assay. <h3>Results</h3> Control-iCM:HCF-MTs (n=23-49), following TGF-β1 stimulation, revealed a shorter contraction duration (p&lt;0.01) and a lower contraction amplitude (p&lt;0.0001) but not altered contraction duration (p=0.5095) and contraction amplitude (p=0.7298) compared to untreated-iCM:HCF-MTs. Interestingly, untreated-SSc-hMDM:iCM:HCF-MTs showed a shorter time-to-peak (p&lt;0.0001), lower beating rate (p&lt;0.01) and higher contraction amplitude (p&lt;0.01) compared to untreated-HC-hMDM:iCM:HCF-MTs (n=41-81, both conditions 3d). Next, TGF-β1-stimulated SSc-hMDM:iCM:HCF-MTs exhibited a lower contraction duration (p&lt;0.01) and contraction amplitude (p&lt;0.0001), shorter relaxation time (p&lt;0.0001), and higher beating rate (p&lt;0.0001) compared to TGF-β1-stimulated HC-hMDM:iCM:HCF-MTs (n=33-42, both conditions 1d). Upon TGF-β1-stimulation, control-iCM:HCF-MTs showed significantly increased profibrotic genes: <i>ACTA2</i> (p&lt;0.001) and <i>COL1A1</i> (p&lt;0.01) (n=4-5), secreted human pro-collagen Iα1 (n=9-10, p&lt;0.0001), and formed visible collagen-rich fibrotic ring. Remarkably, under fibrotic conditions, addition of HC or SSc hMDM into iCM:HCF-MTs resulted in lower mRNA expression of <i>ACTA2</i> (n=4-5, p&lt;0.05, both conditions 7d) and <i>COL1A1</i> (n=4-5, p&lt;0.05, both conditions 7d), lower excretion of human pro-collagen Iα1 protein (n=9-10, p&lt;0.0001, both conditions 7d) and lack of collagen-rich fibrotic ring compared to control-iCM:HCF-MTs. Importantly, independently of culture duration of HC or SSc hMDM, before MT assembly, a similar reduction in profibrotic gene and protein expression was observed. Lastly, the level of apoptosis was similar in all three undifferentiated or TGF-β1-stimulated MTs types. <h3>Conclusion</h3> For the first time, the combination of iCM, HCF and hMDM in a 3D fibrotic cardiac MT model has been established and investigated. Under fibrotic conditions, addition of HC- but not SSc-hMDM into iCM:HCF-MTs influenced MT contractility. Importantly, both HC- and SSc-hMDM reduced TGF-β1-induced fibrotic responses. Scheme 1: Graphical abstract <h3>References</h3> [1]Błyszczuk et al. Cells. 2020. [2]Sala et al. Circ. Res. 2018. <h3>Scheme 1</h3> Graphical abstrac.t. <h3>Disclosure of Interests</h3> Laila Provenzale: None declared, Amela Hukara: None declared, Ievgeniia Kocherova: None declared, Elena Pachera: None declared, Andrea Laimbacher: None declared, Oliver Distler Speakers bureau: Oliver Distler has/had relationships with the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: Bayer, Boehringer Ingelheim, Janssen, Medscape. Consultant of: Oliver Distler has/had relationships with the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: Oliver Distler has/had consultancy relationship with and/or has received research funding from the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three years: Research Grants: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Przemyslaw Blyszczuk: None declared, Gabriela Kania: None declared.